Reversal of hepatitis B virus-induced immune tolerance by an immunostimulatory 3p-HBx-siRNAs in a retinoic acid inducible gene I–dependent manner

Authors

  • Qiuju Han,

    1. Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong University, Jinan, China
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  • Cai Zhang,

    Corresponding author
    1. Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong University, Jinan, China
    • Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan 250012, China
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  • Jian Zhang,

    1. Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong University, Jinan, China
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  • Zhigang Tian

    Corresponding author
    1. Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong University, Jinan, China
    2. Institute of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, China
    • Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan 250012, China
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    • fax: 86-531-8838-3782


  • Potential conflict of interest: Nothing to report.

  • This work was supported by grants from the Natural Science Foundation of China (#90713033), the National 973 Basic Research Program of China (#2007CB815803), and the National 115 Key Project for HBV Research (#2008ZX10002-008).

Abstract

It is extensively accepted that hepatitis B virus (HBV) escapes from innate immunity by inhibiting type I interferon (IFN) production, but efficient intervention to reverse the immune tolerance is still not achieved. Here, we report that 5′-end triphosphate hepatitis B virus X gene (HBx)-RNAs (3p-HBx-short interfering [si]RNAs) exerted significantly stronger inhibitory effects on HBV replication than regular HBx-siRNAs in stably HBV-expressing hepatoplastoma HepG2.2.15 cells through extremely higher expression of type I IFNs, IFN-induced genes and proinflammatory cytokines, and retinoic acid inducible gene I (RIG-I) activation. Also, 3p-HBx-siRNA were more efficient to stimulate type I IFN response than HBx sequence-unrelated 3p-scramble-siRNA in HepG2.2.15 cells, indicating that a stronger immune-stimulating effect may partly result from the reversal of immune tolerance through decreasing HBV load. In RIG-I-overexpressed HepG2.2.15 cells, 3p-HBx-siRNAs exerted stronger inhibitory effects on HBV replication with greater production of type I IFNs; on the contrary, in RIG-I-silenced HepG2.2.15 cells or after blockade of IFN receptor by monoclocnal antibody, inhibitory effect of 3p-HBx-siRNAs on HBV replication was largely attenuated, indicating that immunostimulatory function of 3p-HBx-siRNAs was RIG-I and type I IFN dependent. Moreover, in HBV-carrier mice, 3p-HBx-siRNA more strongly inhibited HBV replication and promoted IFN production than HBx-siRNA in primary HBV+ hepatocytes and, therefore, significantly decreased serum hepatitis B surface antigen and increased serum IFN-β. Conclusion: 3p-HBx-siRNAs may not only directly inhibit HBV replication, but also stimulate innate immunity against HBV, which are both beneficial for the inversion of HBV-induced immune tolerance. (HEPATOLOGY 2011;)

Chronic hepatitis B virus (HBV) infection is closely associated with increased risks of liver cirrhosis and hepatocellular carcinoma. Unfortunately, most chronically HBV-infected patients (CHB) do not benefit from conventional therapy (i.e., interferon-α and lamivudine). Whether HBV infection is cleared or persists as a progressive liver disease is determined by interaction between virus and host factors.1, 2 Although type I interferons (IFNs) can suppress HBV replication, the virus has the counteracting mechanisms to suppress the production and function of IFNs, leading to immune tolerance. For instance, HBV suppresses MxA expression at the promoter level or by inhibiting cellular proteasome activities in an HBV X gene (HBx)-dependent manner.3, 4 Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and even virion particles can directly suppress Toll-like receptor (TLR)-mediated innate immunity in primary hepatocytes by up-regulating anti-inflammatory cytokine transforming growth factor beta (TGF-β).5 HBV polymerase disturbs retinoic acid inducible gene I (RIG-I)- and TLR3-mediated IFN-β induction,6 and HBx protein disrupts the RIG-I pathway by down-regulating mitochondrial antiviral signaling protein.7-9 These findings strongly support the notion that HBV can escape from host innate and adaptive immunity. Thus, determining how to reverse HBV-induced immune tolerance has become an important approach in the treatment of CHB.

It was suggested that decreasing HBV load could potentially benefit patients by reversing virally induced immune tolerance. Recently, the use of RNA interference (RNAi) technology has provided approaches to inhibition of viral gene expression and replication in vitro and in vivo.10 Moreover, it was reported that short interfering RNAs (siRNAs) may trigger innate immunity by “off-target” immunostimulatory effects through the binding and activation of TLRs (e.g., TLR3, 7, and 8), RIG-I, or PKR.11, 12 RIG-I is the key sensor of RNA viruses in the cytosol of cells, recognizing RNA with a 5′-triphosphate group (3p-siRNA) in a sequence-independent manner.13, 14 Although 3p-siRNAs were described to induce type I IFN in several cell types,15-17 other synthetic RIG-I agonists lacking 5′-triphosphate, such as polyinosinic-polycytidylic acid (poly [I:C]),18, 19 and some natural RNAs, such as viral transcripts,13-15 also serve as RIG-I agonists. So, RIG-I activation leads to the production of type I IFNs and inflammatory cytokines, which play an important role in inhibiting HBV replication via reversing HBV-induced tolerance.20

Recently, Hartmann et al. reported on a Bcl2-specific siRNA with 5′-triphosphate ends to silence Bcl2 and simultaneously activate RIG-I. Therapy with such bifunctional 3p-siRNAs revealed strong promotion of apoptosis and inhibition of lung metastasis via activation of innate immune responses by RIG-I as well as silencing of Bcl2.17 Whether this therapeutic strategy with bifunctional siRNA is beneficial for HBV clearance is still unknown. The HBx gene coactivates the transcription of viral and cellular genes and is an ideal target for RNA silencing of chronic HBV infection and HBV-related hepatocellular caicinoma.4 In this study, we compared the ability of immunostimulatory 3p-HBx-siRNAs with regular HBx-siRNAs in inducing anti-HBV innate immunity, in addition to their ability to inhibit HBV replication. Our results demonstrate that 3p-HBx-siRNA has a strong dual function, both on inhibiting HBV replication and on triggering innate immunity in a RIG-I-dependent manner, which synergistically benefits the reversal of HBV-induced immune tolerance.

Abbreviations

APC, antigen-presenting cell; CARD, caspase-recruitment domain; CHB, chronically HBV-infected patients; DCs, dendritic cells; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBV, hepatitis B virus; HBx, hepatitis B virus X gene; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; HBS, Hepes-buffered saline; IFN, interferon; IL, interleukin; IPS-1, IFN promoter stimulator 1; IRF3, interferon regulatory factor 3; ISG, interferon-stimulated gene; LF, Lipofectamine 2000; MDA-5, melanoma differentiation-associated gene 5; MxA, myxovirus resistence protein A; mRNA, messenger RNA; NF-κB, nuclear factor kappa light-chain enhancer of activated B cells; poly(I:C), polyinosinic-polycytidylic acid; PCR, polymerase chain reaction; PRRs, pathogen recognition receptors; RIA, radioimmunoassay; RIG-I, retinoic acid inducible gene I; RNAi, RNA interference; siRNAs, short interfering RNAs; 3p-siRNA, 5′-triphosphate siRNA; SD, standard deviation; TGF, transforming growth factor; TLR, Toll-like receptor; TNF, tumor necrosis factor; Tregs, CD4+CD25+ regulatory T cells.

Materials and Methods

RNA.

We purchased chemically synthesized siRNA from Ribo Company (RiboBio, Guangzhou, China) and GenePharma Company (GenePharma, Shanghai, China). A detailed list of all chemically synthesized siRNAs is provided in Supporting Table 1. In vitro transcribed siRNAs were synthesized by using the Ambion (Austin, TX) MEGAscript kit and the In Vitro Transcription T7 Kit (Takara, Dalian, China) (for DNA templates, see Supporting Table 2).

Plasmids.

Plasmid expressing full-length human Flag-RIG-I was kindly provided by Takashi Fujita (Kyoto University, Kyoto, Japan). Plasmid expressing caspase-recruitment domain (CARD) domains of RIG-I was kindly provided by Ju-Tao Guo (Drexel University College of Medicine, Philadelphia, PA).

Cell Lines and Cell Culture.

HepG2 (maintained in our lab) were grown in RPMI-1640 medium (Gibco BRL, Gaithersburg, MD), supplemented with 10% fetal bovine serum (FBS). HepG2.2.15 cells (serotype ayw, genotype D), which were derived from HepG2 cells transfected with a plasmid carrying two head-to-tail copies of HBV genome DNA, were maintained in complete Dulbecco's modified Eagle's medium (Gibco), supplemented with 10% FBS. All cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2.

Transfection.

HepG2.2.15, HepG2 cells, or primary mouse hepatocytes were transfected with poly I:C (Sigma-Aldrich, St. Louis, MO), chemically synthesized siRNAs, or in vitro transcribed siRNAs with Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. The siRNAs were prepared at the concentrations of 50 or 100 nM. The final concentration of poly I:C was 50 μg/mL.

Quantitative Real-Time Polymerase Chain Reaction Analysis.

Levels of target genes were normalized to that of β-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (for details, see Supporting Information).

Analysis of HBV DNA.

Viral particles in supernatants were quantified by real-time polymerase chain reaction (PCR), according to instructions of the FQ-PCR HBV-DNA kit (Da-An, Guangzhou, China) (see Supporting Information).

Animal Study.

The pAAV/HBV1.2 vector was delivered into C57BL/6 mice using the hydrodynamic tail vein injection method. Four weeks later, RNAs were injected every other day three times after complexation with N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP; Roche, Indianapolis, IN), according to the manufacturer's protocol. Briefly, for each mouse, we injected with 200 μL containing 50 μg of chemically synthesized or in vitro transcribed siRNAs with or without DOTAP complexation: 30 μL of DOTAP were mixed with 50 μg of siRNA in 170 μL of Hepes-buffered saline (HBS), incubated for 20 minutes, and subsequently injected intravenously in the retro-orbital vein. Control mice received HBS and DOTAP. On the following day of last injection, mice were sacrificed, then HBsAg and IFN-β in sera were assayed by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), respectively.

Immunocytochemical Staining.

Intrahepatic HBcAg was visualized by immunohistochemical staining of tissues by rabbit anti-HBcAg antibody (GeneTech, Shanghai, China) and Envision System, horseradish peroxidase (GeneTech, Shanghai, China). Liver sections were also stained with hematoxylin.

ELISA.

Levels of HBsAg and HBeAg in culture supernatants were detected by using an ELISA kit from RongSheng (Shanghai, China). HBeAg is expressed as National Center Units/mL. A mouse IFN-β ELISA kit was obtained from PBL Biomedical Laboratories (Piscataway, NJ).

RIA.

Levels of HBsAg and HBeAg in serum were detected by using an RIA kit, according to the manufacturer's instructions (Beijing North Institute of Biological Technology, Beijing, China).

Analysis of isolation and culture of primary mouse hepatocytes, western blotting analysis, ELISA, and luciferase reporter gene assay are included in the Supporting Information.

Statistical Analysis.

Statistical analysis was performed using a paired Student's t-test. A value of P < 0.05 was considered statistically significant.

Results

HBV-Transfected HepG2.2.15 Cells Were Immunotolerant to Poly I:C Stimulation.

It has been reported that HBV inhibits the innate immune response in liver cells, including suppression of type I IFN production and IFN-stimulated gene induction.5 By comparison, in the expression of type I IFN and innate immune-related genes in virus-free HepG2 cells and HBV-infected HepG2.2.15 cells, we found that the expression levels of IFN-α, IFN-β, interferon-stimulated gene (ISG)15, myxovirus resistance protein A (MxA), and RIG-I were extremely lower in HepG2.2.15 than those in HepG2 cells (Supporting Fig. 1A). However, the expression level of the immunosuppressive cytokine, TGF-β, in HepG22.2.15 was higher than that in HepG2 cells, whereas the interleukin (IL)-10 expression level was not significantly different between the two cell lines (data not shown), indicating that the presence of HBV in the HepG2.2.15 cells inhibited the expression of innate immune-related genes, and the high expression of TGF-β also contributed to the immune tolerance caused by HBV. To confirm this, HepG2 and HepG2.2.15 cells were stimulated with intracellular poly I:C, which might activate innate immune response through the RIG-I recognition pathway, as previously reported.6 In this experiment, although the poly I:C treatment strongly stimulated IFN responses, showing the increased messenger RNA (mRNA) expressions of IFN-α, IFN-β, ISG15, and MxA in both cells, HBV-transfected HepG2.2.15 cells were in a much lower innate immune response to stimulation with poly I:C, if compared with HepG2 cells (Supporting Fig. 1B). In accord with previous reports,6 these results further demonstrated that HBV in HepG2.2.15 cells suppressed the RIG-I-mediated innate immune responses as a critical mechanism to escape from intrinsic innate immunity.

5′-Triphosphate siRNA Exerted a Strong Immunostimulatory Effect in a Sequence-Independent Manner in HBV-Transfected HepG2.2.15 Cells.

Previous studies have shown that short 5′-triphosphate RNAs by in vitro transcription are potent RIG-I ligands that induce the production of IFNs.15, 17, 21-23 Here, we observed that HBV RNA sequence-unrelated 3p-scramble-siRNA or 3p-GAPDH-siRNA transfection significantly increased the expression of RIG-I mRNA in HepG2.2.15 cells after 24 hours at the concentration of 100 nM by 6.4- or 11.8-fold, respectively (Supporting Fig. 2A), and was followed by a significant increase in the induction of IFN-α and IFN-β (Fig. 1A,B). More interestingly, 3p-scramble-siRNA and 3p-GAPDH-siRNA also inhibited the mRNA expression of HBx and HBs/p and even decreased supernatant HBsAg and HBeAg content in the culture of HepG2.2.15 cells (Fig. 2), suggesting that the activation of innate immune responses by 3p-scramble-siRNA or 3p-GAPDH-siRNA might partially inhibit HBV replication through the RNAi-unrelated, but intrinsic, innate immune pathway.

Figure 1.

Immunostimulatory 3p-HBx-siRNA improved the inversion of HBV-induced innate immune tolerance. (A,B) Chemically synthesized scramble siRNA (100 nM) and HBx-siRNA (siRNA), or the corresponding in vitro transcribed 3p-scramble siRNA (100 nM) and 3p-HBx-siRNA (3p-siRNA), were transfected into HepG2.2.15 cells. The mRNA expression level of type I IFN was determined by quantitative real-time PCR. (C) Level of RIG-I was determined by quantitative real-time PCR and western blotting. (D) HepG2.2.15 cells were transfected with synthetic scramble siRNA, HBx-siRNA, 3p-scramble, 3p-HBx-siRNA3, or their combinations for 24 hours. The final concentration of siRNA was 100 nM. mRNA expressions of IFN-α and IFN-β were analyzed by quantitative real-time PCR. Data are represented as the mean ± standard deviation (SD) from at least three independent experiments. #P < 0.05: 3p-Scramble-siRNA or 3p-GAPDH versus LF; *P < 0.05: 3p-HBx-siRNA versus HBx-siRNA.

Figure 2.

Immunostimulatory 3p-HBx-siRNA exerted stronger inhibitory effect on HBV replication. Chemically synthesized HBx-siRNA (siRNA) or the corresponding in vitro transcribed 3p-HBx-siRNA (3p-siRNA) or 3p-scramble-siRNA or 3p-GAPDH (100 nM) were transfected into HepG2.2.15 cells by using LF. LF alone was used as a control. mRNA expression levels of HBx (A) and HBs/p (B) genes were detected by quantitative real-time PCR. The level of each sample is expressed as the percentage of the RNA level in LF-treated cells. Supernatants were harvested after 48 hours, and levels of HBsAg (C) and HBeAg (D) were detected using ELISA. Data are expressed as the mean ± SD from at least three independent experiments. #P < 0.05: 3p-Scramble-siRNA or 3p-GAPDH versus LF; *P < 0.05: 3p-HBx-siRNA versus HBx-siRNA.

Meanwhile, regular HBx-siRNAs, with weak immunostimulatory function, were chemically synthesized (HBx-siRNA1, HBx-siRNA2, and HBx-siRNA3) and transfected into HepG2.2.15 cells at the concentration of 100 nM. As expected, chemically synthesized HBx-siRNAs significantly inhibited HBx and HBs/p mRNA expression (P < 0.05, compared to Lipofectamine 2000 (LF)] at 24 hours post-transfection, and the secretion of HBsAg and HBeAg was significantly decreased at 48 hours (Fig. 2), with a weak induction of RIG-I (Supporting Fig. 2B) and type I IFN response (Fig. 1A,B). We proposed that the weakly increased levels of RIG-I mRNA and type I IFN mRNA might, at least partially, result from the attenuation of HBV-induced immunotolerance after decreasing HBV virus load by the inhibition of HBV replication.

We then combined the immunostimulatory functions of 3p-scramble and HBx-silencing function of HBx-siRNA by producing 3p-HBx-siRNA. We observed that all three 3p-HBx-siRNAs obviously activated innate immune responses by markedly increasing IFN-β promoter function (Supporting Fig. 3A), type I IFN production (Fig. 1; Supporting Fig. 3B), and inflammatory secretion (Supporting Fig. 4A), as well as obviously decreasing the production of immunosuppressive cytokines (Supporting Fig. 4B). We also observed that RIG-I expression was augmented, and the expressions of type I IFN-induced genes (MxA and ISG15) were increased at 48 hours post–3p-HBx-siRNAs transfection (Supporting Fig. 5A). Additionally, 3p-HBx-siRNA transfection promoted the degradation of NF-kappa-B inhibitor alpha (IκBα) at 30 minutes, reaching its maximum at 90 minutes, and returning to a lower level at 180 minutes (Supporting Fig. 5B), and augmented the phosphorylation of p44/42 extracellular signal-regulated kinase (Supporting Fig. 5C). Together, these results suggested that the transfection of 3p-HBx-siRNAs potently activate RIG-I signaling and thus induce the production of type I IFN and proinflammatory cytokines in hepatocytes.

Immunostimulatory 3p-HBx-siRNA Promoted the Inversion of HBV-Induced Innate Immune Tolerance.

After observing the immunostimulatory function of triphosphate siRNA (e.g., 3p-scramble siRNA or 3p-HBx-siRNA), and HBx-siRNA's direct inhibition of HBV replication (e.g., HBx-siRNA or 3p-HBx-siRNA), we then started to explore the roles of the dual function of 3p-HBx-siRNAs in reversing HBV-induced immunotolerance. First, we compared the effects of chemically synthesized HBx-siRNA with that of in vitro transcribed 3p-HBx-siRNAs on HBV replication. Though both HBx-siRNA3 and 3p-HBx-siRNA3 exerted a significant inhibitory effect on HBV replication, 3p-HBx-siRNA (3p-siRNA3) was more effective in inhibiting HBV replication than HBx-siRNA, showing significantly lower expression of HBx and HBs/p mRNA of cells (Fig. 2A,B) at 24 hours post-transfection. Importantly, treatment with 3p-HBx-siRNAs resulted in a greater reduction in HBsAg and HBeAg at 48 hours (Fig. 2C,D). By western bloting analysis, we also observed the stronger silencing effect of 3p-HBx-siRNAs on HBx expression at the protein level (Supporting Fig. 6A), and the level of intracellular HBcAg was also decreased significantly by immunohistochemical staining (Supporting Fig. 6B). So, 3p-HBx-siRNA absolutely exerted a more inhibitory effect on HBV replication, in addition to its direct HBx silencing.

Second, we compared the effects of chemically synthesized HBx-siRNAs with that of in vitro transcribed 3p-HBX-siRNAs on RIG-I activation. As expected, both immunostimulatory 3p-HBx-siRNAs and 3p-scramble-siRNA induced higher levels of RIG-I than HBx-siRNA in HBV-transfected HepG2.2.15 cells at both the mRNA and protein level (Fig. 1C). To determine whether 3p-HBx-siRNA would reverse HBV-induced immunotolerance by its dual functions, we examined type I IFN production by observing a variety of treatment combinations in experiment groups, according to immunostimulating and HBV silencing effects. It was noted that 3p-scramble exerted a similar effect on type I IFN production as 3p-scramble+ scramble, indicating scramble-siRNA itself exerted a very weak stimulation to RIG-I. Although the 3p-scramble alone strongly stimulated IFN responses, responses were more efficient when the HBx gene was silenced in the 3p-scramble+HBx-siRNA group (Fig. 1D). Meanwhile, 3p-scramble+HBx-siRNA showed a stronger stimulating effect on type I IFN production than scramble+HBx-siRNA, indicating that HBx-siRNA itself exerted a weak effect on IFN production. 3p-HBx-siRNA alone had a mostly strong stimulation on IFN production, a little higher than 3p-scramble+HBx-siRNA, demonstrating that 3p-HBx-siRNA has a dual function that is beneficial to reversing of HBV-induced immunotolerance (Fig. 1D).

Third, to further determine whether 3p-HBx-siRNA would reverse HBV-induced immunotolerance, we compared the innate immune response of HepG2 cells with that of HBV-transfected HepG2.2.15 cells after stimulation with immunostimulatory HBV RNA sequence-unrelated siRNA (e.g., 3p-scramble siRNA) or immunostimulatory HBx siRNA (e.g., 3p-HBx-siRNA), respectively. We found that both 3p-HBx-siRNAs and 3p-scramble-siRNA equally strongly stimulated the production of type I IFN (Fig. 3A,B) and type I IFN downstream proteins (ISG15 and MxA) (Fig. 3C,D) in HepG2 cells; however, in HepG2.2.15 cells, 3p-HBx-siRNAs were more efficient at stimulating the production of type I IFN, ISG15, and MxA than 3p-scramble-siRNA (Fig. 3), demonstrating that 3p-HBx-siRNA exerted a stronger immunostimulating effect because of their ability to reverse HBV-induced immunotolerance by decreasing HBV load in HepG2.2.15 cells. Therefore, our results demonstrated that treatment with 3p-HBx-siRNAs not only inhibits HBV replication, but also stimulates innate immune responses in HepG2.2.15 cells and, therefore, dramatically reverses HBV-induced immune tolerance.

Figure 3.

Comparison of immunostimulatory function of 3p-HBx-siRNA in HepG2 cells and HepG2.2.15 cells. 3p-scramble siRNA (3p-scramble) and 3p-HBx-siRNA were transfected into HepG2 or HepG2.2.15 cells for 24 hours, respectively. mRNA expressions of IFN-α (A), IFN-β (B), MxA (C), and ISG15 (D) were analyzed by quantitative real-time PCR. Changes in HepG2 cells and HepG2.2.15 cells were compared. Data are represented as the mean ± SD from at least three independent experiments. *P < 0.05: versus LF-treated group.

Immunostimulatory Function of 3p-HBx-siRNA Correlates to RIG-I-Dependent Type I IFN Production.

To further determine whether the 3p-HBx-siRNAs, indeed, stimulated innate immune response through the activation of RIG-I and, subsequently, contributed to HBV inhibition, we first transfected HepG2.2.15 cells with a plasmid containing the caspase-recruitment domain of RIG-I, followed by stimulation with 3p-HBx-siRNAs, and then assessed the changes of HBV expression. The results showed that overexpression of the CARD domain of RIG-I markedly promoted the inhibition of HBV DNA replication (Fig. 4A), HBx mRNA expression (Fig. 4B), and contents of HBsAg (Fig. 4C) and HBeAg (Fig. 4D) by 3p-HBx-siRNAs. Importantly, overexpression of the CARD domain of RIG-I significantly promoted the production of type I IFNs (Fig. 4E,F). These results suggested that overexpression of RIG-I further promoted the inhibition of HBV replication by 3p-HBx-siRNAs via activation of the RIG-I pathway.

Figure 4.

RIG-I overexpression enhanced 3p-HBx-siRNA-mediated HBV inhibition. HepG2.2.15 cells were transfected with 2 μg of RIG-I/CARD plasmid or the control plasmid. Twenty-four hours later, cells were transfected with 3p-HBx-siRNA (50 nM). (A) HBV DNA copy number was analyzed by quantitative PCR. (B) mRNA expression level of HBx was measured by quantitative real-time PCR at 24 hours. (C,D) The secreting protein levels of HBsAg and HBeAg in supernatants were examined by ELISA. (E,F) mRNA expressions of IFN-α and IFN-β were examined by quantitative real-time PCR. Levels of examined parameters were represented as percentages to the level of LF-treated control-vector transfected cells. Data are expressed as the mean ± SD from at least three independent experiments. *P < 0.05: versus LF-treated group.

We further knocked down RIG-I expression in HepG2.2.15 cells by RIG-I RNAi (Fig. 5A), and found that mRNA expression (Fig. 5B), supernatant contents of HBsAg and HBeAg (Fig. 5D,E), and HBx mRNA expression (Fig. 5C) automatically increased after the silencing of RIG-I. We then stimulated these RIG-I-low cells with 3p-HBx-siRNAs, and observed that RIG-I knockdown strongly attenuated the silencing effect of 3p-HBx-siRNAs (Fig. 5C). These results suggest that RIG-I activation correlates with the stronger inhibition of HBV replication by 3p-HBx-siRNAs, and overexpression of RIG-I promotes antiviral effects by 3p-HBx-siRNAs.

Figure 5.

RIG-I RNAi attenuated 3p-HBx-siRNA-induced HBV inhibition. HepG2.2.15 cells were transfected with RIG-I-specific siRNA for 24 hours, and mRNA levels of RIG-I (A) as well as HBsAg and HBeAg (B) were detected by quantitative real-time PCR. The level was expressed as percent of level in scramble siRNA-treated cells. Data are expressed as the mean ± SD from at least three independent experiments. *P < 0.05: versus scramble siRNA-treated cells. After transfected with RIG-I-specific siRNA for 24 hours, HepG2.2.15 cells were further transfected with 3p-HBx-siRNA (50 nM) for another 24 hours. The level of HBx mRNA (C) of cell extracts and the protein contents of HBsAg (D) and HBeAg (E) in supernatant were examined as described before. Levels are expressed as percent of level in LF-treated cells. Data are expressed as the mean ± SD from at least three independent experiments. *P < 0.05: versus LF-treated group.

To demonstrate the role of type I IFN in 3p-HBx-siRNA-mediated dual inhibition of HBV replication in HepG2.2.15 cells, we first detected the expression of IFNR1 in HepG2 and HepG2.2.15 cells by real-time PCR, and found that the expression level was not different between these two cells (Fig. 6A). To determine whether IFN induction is involved in HBV-induced immune tolerance and RIG-I-mediated HBV inhibition, we blocked the type I IFN pathway with a neutralizing antibody against the IFN-α/β receptor for 12 hours, then transfected the cells with 3p-HBx-siRNAs for 24 hours. The results demonstrated that the blockade of IFN receptors significantly attenuated the inhibitory effect of 3p-HBx-siRNAs on the expression of HBx mRNA (Fig. 6B) and the secretion of HBsAg (Fig. 6C) and HBeAg (Fig. 6D). These results suggested that 3p-HBx-siRNAs-induced innate immune responses (especially the production of type I IFN) play important roles in HBV inhibition and clearance, in addition to a direct RNA-silencing effect.

Figure 6.

Inhibitory effect of immunostimulatory 3p-HBx-siRNA on HBV replication was attenuated by type I IFN receptor neutralization. (A) mRNA levels of IFNR1 in HepG2 and HepG2.2.15 cells by real-time PCR analysis. Histogram represents the relative expression level of IFNR1 after normalization to its corresponding internal control, β-actin. (B) HepG2.2.15 cells were treated with IFNα/β receptor-blocking monoclonal antibody or isotype control for 12 hours before 3p-HBx-siRNA transfection. Twenty-four hours after transfection, the HBx mRNA level was examined. (C,D) supernatant HBsAg and HBeAg contents were measured by ELISA. Levels are expressed as percentage to isotype control-treated cells. Data are expressed as the mean ± SD from at least three independent experiments. *P < 0.05: versus respective isotype control treatment group.

Immunostimulatory 3p-HBx-siRNA Exerted a Stronger Inhibitory Effect on HBV Replication in HBV-Carrier Mice.

Because the conclusion that immunostimulatory 3p-HBx-siRNA exerted a stronger inhibitory effect on HBV replication came from an HBV-infected cell line (HepG2.2.15), we then confirmed the conclusion by in vivo experiments. First, C57BL/6 mice were hydrodynamically injected with HBV-containing plasmid by the tail vein, and HBV-carrier mice were obtained as described in Patients and Methods. The fresh mouse hepatocytes were then isolated and transfected with HBx-siRNA and 3p-HBx-siRNAs, respectively. Treatment with 3p-HBx-siRNAs (50 nM) resulted in a significantly greater reduction of HBx, HBs/p, and HBc/p expression (Fig. 7A) and, also, significantly higher mRNA levels of IFN-α, IFN-β, and RIG-I (Fig. 7B) than HBx-siRNA, indicating that 3p-HBx-siRNA exerts stronger inhibitory effects on HBV replication and promotion of type I IFN production than HBx-siRNA in primary murine HBV+ hepatocytes. Second, we also examined the in vivo antiviral activity of 3p-HBx-siRNA and HBx-siRNA in HBV-carrier mice. We treated HBV-carrier mice with 3p-HBx-siRNA or HBx-siRNA on days 3, 6, and 9, respectively. It was noted that the levels of HBsAg in serum (Fig. 7C) and HBcAg in hepatocytes (Fig. 7D) were significantly decreased, and that serum IFN-β was significantly increased (Fig. 7E) in HBV-carrier mice by 3p-HBx-siRNA treatment than that by the HBx-siRNA-treated group. These results suggested that 3p-HBx-siRNAs not only exert HBV-specific gene-silencing effects, but also reversed HBV-induced immune tolerance, therefore revealing a stronger anti-HBV activity in vivo.

Figure 7.

Immunostimulatory 3p-HBx-siRNA exerted a stronger inhibitory effect on HBV replication in HBV-carrier mice. (A,B) HBV-inhibiting and -immunostimulatory effects of 3p-HBx-siRNA on freshly isolated primary HBV-containing hepatocytes. Primary mouse hepatocytes were isolated from HBV-carrier mice, then transfected with LF, HBx-siRNA, or 3p-HBx-siRNA, respectively. Twenty-four hours after transfection, mRNA expression levels of HBx, HBs/p, and HBc/p (A) and IFN-α, IFN-β, and RIG-I (B) were examined by quantitative real-time PCR. The level of each sample is expressed as the percentage of RNA level in LF-treated cells. (C,D) HBV-inhibiting and -immunostimulatory effects of 3p-HBx-siRNA in HBV-carrier mice. Three days after HBx-siRNA or 3p-HBx-siRNA hydrodynamic tail vein injection, HBV-carrier mice were sacrificed and the serum contents of HBsAg (C), immunohistochemical staining for HBcAg (original magnification: ×400) (D), or serum IFN-β level (E) were measured. Data are expressed as the mean ± SD from at least three independent experiments. *P < 0.05: versus siRNA-treated group.

Discussion

Our results (as summarized in Fig. 8) demonstrate that transfection with 3p-HBx-siRNAs inhibited HBV replication not only by direct silencing of HBx expression, but also by enhancing type I IFN production and inflammatory cytokines through RIG-I activation, and the adjuvant effects of inflammatory cytokines could potentially prime adaptive immunity against HBV. On the one hand, in synergy with direct HBx silencing, siRNA-induced activation of innate immunity would further promote the HBx-specific, siRNA-mediated inhibition of HBV replication; on the other hand, 3p-HBx-siRNA-mediated reduction of the HBV load would be helpful in attenuating the HBV-induced immune tolerance by increasing the production and function of type I IFNs. In addition, 3p-HBx-siRNA-induced activation of RIG-I can also result in the enhanced production of proinflammatory cytokines (such as TNF-α, IL-6, and IL-8) and the decreased production of immunoinhibitory cytokines (TGF-β and IL-10). Therefore, the decrease in HBV load, the increase in activation and function of type I IFNs, and the priming of the anti-HBV adaptive immunity may finally benefit toward the reversal of HBV-induced immune tolerance.

Figure 8.

Working model of 3p-HBx-siRNA-induced RIG-I-mediated anti-HBV innate immunity. (1) Direct silence of HBV by 3p-HBx-siRNA. (2) 3p-HBx-siRNA-induced RIG-I-mediated anti-HBV innate immunity via production of type I IFNs and induction of antivirus proteins by type I IFN signaling. (3) Inhibition of RIG-I activation and signaling by HBV. (4) 3p-HBx-siRNA-induced RIG-I-mediated anti-HBV adaptive immunity via production of proinflammatory cytokines as immune adjuvant.

The natural history of HBV infection and progression of clinical disease are mediated through complicated interactions between virus and host immune response. Whether HBV infection is cleared or persists as a progressive liver disease is determined by both viral and host factors.1, 2 It has been demonstrated that activation of pathogen recognition receptors (PRRs), including TLRs, RIG-I, and melanoma differentiation-associated gene 5 (MDA-5), can induce the inhibition of HBV replication via innate immune responses.20 The local innate immune responses of the liver play major roles in inhibiting HBV replication, especially through the production of large amounts of antiviral cytokines (particularly type I IFN).24 However, HBV develops strategies to suppress antiviral immune response. It has been reported that in CHB patients, HBV-specific immune responses are either absent or low, with deficiency of innate and adaptive immune cells and reduced expression of TLRs.25 HBV has also been shown to suppress TLR-mediated innate immune responses in parenchymal and nonparenchymal liver cells.5 In this study, we observed that the expressions of innate immune receptor RIG-I, type I IFNs, and IFNs-inducible antiviral genes were all lower in HBV-transfected HepG2.2.15 cells than those in virus-free HepG2 cells (Supporting Fig. 1A), and the innate immune response to poly I:C stimulation was much lower in HepG2.2.15 cells (Supporting Fig. 1B). HBx-siRNAs have an obvious HBx silencing effect and weak inducing ability of RIG-I activation, while 3p-scramble siRNAs have no HBV inhibition effect, but strong ability of RIG-I activation due to their 5′-triphosphate structure. We then synthesized 3p-HBx-siRNAs, which, we hoped, would be a dual-functional molecule. In addition to its extraordinary stimulation of innate immunity (Fig. 1; Supporting Figs. 2-5), 3p-HBx-siRNA was the strongest inhibitor of HBV replication in our experiments (Fig. 2). The dual function was also confirmed in primary mouse HBV+ hepatocytes and the in vivo HBV-carrier mouse, showing that 3p-HBx-siRNA exerted a stronger inhibitory effect on HBV replication and induced a higher level of IFN-β in serum (Fig. 7). Interestingly, in our study, though both 3p-HBx-siRNAs and sequence-independent 3p-scramble-siRNA equally stimulated type I IFN response in virus-free HepG2 cells, 3p-HBx-siRNAs were more efficient at stimulating type I IFN response in HepG2.2.15 cells than 3p-scramble-siRNA (Fig. 1), possibly because of a reversal of HBV-induced immune tolerance after directly decreasing HBV load by 3p-HBx-siRNA. Importantly, though 3p-scramble-siRNA stimulates IFNs responses, the responses became more efficient when the HBx gene was silenced by 3p-scramble-siRNA+HBx-siRNA (Fig. 1D), a combination with similar function to 3p-HBx-siRNA alone, confirming that 3p-HBx-siRNA has a dual function beneficial to synergistically reverse HBV-induced immune tolerance.

The immune tolerance caused by HBV infection is characterized by an immunosuppressed environment in the liver with functional impairment of dendritic cells (DCs), T, and NK cells, an abnormal increase of CD4+CD25+ regulatory cells (Tregs), and imbalance of cytokines.1 The immunosuppressive cytokines, TGF-β and IL-10, play key roles in liver immune tolerance,26 wheras Th1 cytokines and proinflammatory cytokines, such as TNF-α, play critical roles in promoting adaptive immune responses. Our data showed that TGF-β was significantly enhanced in HBV-transfected HepG2.2.15 cells, compared with that in noninfected HepG2 cells (Supporting Fig. 1). 3p-HBx-siRNA treatment significantly reversed the imbalance by suppressing TGF-β and IL-10 production while promoting expressions of TNF-α, IL-6, and IL-8 (Supporting Fig. 4). We hypothesized that RIG-I-mediated activation of innate immune responses and the correction of imbalance of cytokines may further enhance the function of DCs and weaken the suppressive activity of Tregs, which could possibly facilitate adaptive immune responses and, finally, help to break the HBV-induced immune tolerance. Accordingly, Anz et al. recently showed that activation of RIG-I and MDA-5 by RNA viruses blocked the suppressive function of Tregs in a direct, antigen-presenting cell (APC)-independent manner.27 The exact mechanism in this study will be further investigated.

The mechanism of HBV inhibiting RIG-I activation is still not totally defined. It is conceivable that the HBV virus may directly inhibit RIG-I activation, a similar mechanism to their effect on TLRs.5 Alternatively, HBV may suppress the activation of signaling molecules downstream of RIG-I activation, such as IFN promoter stimulator 1 (IPS-1), IFN regulatory factor 3 (IRF-3), or NF-κB, leading to down-regulation of type I IFN production and reduced antiviral activity. In keeping with this hypotheses, recent findings revealed that HBV polymerase and HBx can disturb the RIG-I pathway by interfering with IRF3 or down-regulating IPS-1.6-9 However, the exact mechanism needs to be further clarified. RIG-I recognizes RNA viruses, 5′-triphosphate RNA, or short, blunt, double-stranded RNA in the cytosol of cells.15-19, 22 In view of the important role of RIG-I activation, the simultaneous combination of gene silencing and the activation of innate immune responses may be beneficial for the treatment of cancer and other viral infectious diseases, as explored in studies by Hartmann and Sioud in an antitumor model.28 In the present study, we designed the 3p-HBx-siRNAs to exhibit two distinct functional properties: HBx gene silencing and RIG-I activation. These properties may cooperate to promote the inhibition of HBV. To our knowledge, we are the first to demonstrate the potential role of bifunctional 3p-siRNAs in the treatment of HBV infection. Moreover, this therapeutic strategy can be extended to other viral diseases, especially for positive- and negative-strand RNA viruses (e.g., influenza virus, rabies virus, or hepatitis C virus), which are recognized by RIG-I.13, 14, 19, 22

It is not certain whether immune cells, such as Kupffer cells, hepatic DC cells, and even liver sinusoid endothelial cells, in addition to hepatocytes within the liver, might either take up siRNAs or respond to immune stimulations induced by siRNA, then activate to recruit other immune cells into the liver to mount an adaptive immune response. It should also be noticed that inducing potent type I IFN responses that can activate a wide range of innate and adaptive immune cells would potentially have serious adverse effects, particularly if the therapy is administered systemically. Thus, the specifically targeted delivery of immunostimulatory siRNAs into the liver may stimulate antiviral immunity more effectively, with fewer side effects than with systemic delivery. In conclusion, bifunctional siRNAs with both gene-silencing and innate immune-activation properties represent a new potential strategy for the treatment of viral infection.

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